The Ras oncogene is a causative agent in about one-third of all human cancers, and there are no effective drug-based treatments available that specifically target the oncogenic Ras protein. We detected an activity in a crude E. coli extract that efficiently converts GTP-bound oncogenic Ras proteins to an inactive GDP bound form. The activity was purified and a single protein responsible for the activity was identified as nucleoside diphosphate kinase (NDK). We find that NDK can specifically inactivate several oncogenic Ras proteins, but not wild-type Ras, in vitro and in vivo. We propose to extend these findings to test the feasibility of biochemically inactivating oncogenic Ras in vivo as a means of inhibiting tumorigenesis. The Specific Aims of the proposed studies are: I. To establish the biochemical mechanism by which NDK promotes the specific inactivation of oncogenic forms of the Ras GTPase. This is expected to provide insights into the biochemical defect associated with oncogenic Ras proteins and potential strategies for promoting specific Ras inactivation. II. To assess the ability of NDK to antagonize the oncogenic actions of Ras in cell culture transformation assays and in a mouse model of K-Ras-induced tumors. Here, NDK will be utilized as a tool for examining the feasibility of promoting biochemical inactivation of Ras in vivo as a therapeutic strategy. III. To test the hypothesis that selective loss of NM23 (the human ortholog of NDK, which has been identified previously as a tumor/metastasis suppressor of unknown function) in human tumors is associated with increased potency of oncogenic forms of Ras. IV. To utilize a high throughput biochemical screening strategy to identify small molecules with the potential to facilitate inactivation of oncogenic forms of Ras. This will be performed in close collaboration with the Harvard Institute of Chemistry and Chemical Biology. Together, the proposed studies are expected to lead to the eventual development of a novel therapeutic approach for achieving specific targeting of human tumors that harbor oncogenic mutant forms of Ras.